FIG. 2.

Mig3 is phosphorylated during genotoxic stress. CDY61 cells expressing Mig3-Myc were grown in YPD (lanes labeled Control, No MMS, No UV, and No HU) or treated with 5 μg of phleomycin per ml for 30 min (Phleomycin), 200 μM cadmium for 1 h (Cd), 200 mM HU for 2 h, or 1% MMS for 30 min followed by neutralization with an excess of sodium thiosulfate (MMS). For UV irradiation (UV), cells were concentrated and spread on the surface of a YPD plate that was then irradiated with 80 J of 254-nm light per m² in a Stratagene cross-linker. Cells were resuspended for an additional hour in YPD before TCA protein extraction and immunoblotting. (B) Protein extracts were prepared from cells grown in YPD or in YPD containing 200 mM HU for 2 h. The extracts were incubated with or without 10 U of CIP. Phosphatase inhibitors were added to some reactions as a control showing that the mobility shifts were due to the action of the CIP. (C) Time course of Mig3 phosphorylation after the addition of 200 mM HU to CDY61 cells growing exponentially in YPD. The position of an 80-kDa molecular mass marker is shown next to the immunoblots.